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Establishment of large scale-recombinant human TPO preparation, its clinical application and determination of its activity site

Research Project

Project/Area Number 01480293
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 内分泌・代謝学
Research InstitutionMiyazaki medical College

Principal Investigator

OHTAKI Sachiya  Miyazaki Medical College, Laboratory for Clinical Investigation, Professor, 医学部, 教授 (00001917)

Co-Investigator(Kenkyū-buntansha) KOTANI Tomio  Miyazaki Medical College, Laboratory for Clinical Investigation, Lecture, 医学部, 講師 (10161936)
Project Period (FY) 1989 – 1990
Project Status Completed (Fiscal Year 1990)
Budget Amount *help
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1989: ¥5,000,000 (Direct Cost: ¥5,000,000)
KeywordsHuman TPO / Recombinant TPO / Measurement of autoantibodies / Enzyme activity / 甲状腺ペルオキシダ-ゼ / 抗体定量 / 酵素活性部位 / 臨床応用 / 構造
Research Abstract

In order to prepare a large amount of recombinant human TPO (rhTPO), we constructed an expression plasmid, pSV2-HTPO. dhfr, containing both human thyroid peroxidase and mouse dihydrofolate reductase cDNAs and transfected into chinese hamster ovary cells. A subline producing a large amount of rhTPO was established by selecting clones resistant to methotrexate. The molecular weight of the rhTPO was the same as purified hTPO (native TPO). This expressed protein had peroxidase activity when determined by guaiacol oxidation.
When we compared immunoreactivity of rhTPO with that of native TPO using competitive binding inhibition and dilution tests in micro-ELISA system, rhTPO had almost the same immunoreactivity as native hTPO. In respect to background absorbance, rhTPO was superior to native hTPO. Since international standard serum for anti-TPO autoantibodies was introduced into this micro-ELISA system using rhTPO as antigen to measure anti-TPO autoantibodies, we have been able to express ant … More i-TPO autoantibody concentration as IU/ml. Using about one hundred sera from patients with thyroid-related disorders, we examined the correlation between titer of microsome test and anti-TPO antibody concentration. In the sera without anti-thyroglobulin in autoantibodies (less tha X100 in thyroid test), a significant correlation was observed between them, although no correlation was calculated using all sera. The above result was consistent with our theme that a kit measuring autoantibodies specific for TPO should be developed.
To study His residue binding to heme iron, we expressed both hTPO・cDNAs, longer cDNA consisting of 3,048 nucleotides and shorter cDNA lacking the 10th exon. The former cDNA showed both hTPO protein and TPO activity, when it was introduced to vaccinia virus expression system. The latter cDNA, however, could not express TPO activity. From the above results, we considered two His residues contained in the 8th and 10th exons as proximal and distal His residues, respectively, among three His residues belonging to three respective exons, the 8th, 9th, and 10th. However, a possibility that conformationl change in shorter cDNA expression might disturb TPO activity expression remains to be solved. Less

Report

(3 results)
  • 1990 Annual Research Report   Final Research Report Summary
  • 1989 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Shioko Kimura: "cDNAーdirected expression of human thyroid peroxidase" FEBS LETT.250. 377-380 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Junichiro Hata: "Stable high level expression of human thyroid peroxidase in cultured chinese hamster ovary cells." Biochem.Biophys.Res.Commun.164. 1268-1273 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Satoru Yagihashi: "Characterization and application of recombinant human thyroid peroxidase expressed in cultured chinese hamster ovary cells" J.Immunol.Methods.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Shinya Yamashita: "Secretion of membrane binding protein, human thyoid peroxidase, expressed in mammalian cells" J.Endocrinol.

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Kimura, S., Kotani, T., Ohtaki, S. and Aoyama, T.: "cDNA-directed expression of human thyroid peroxidase" FEBS Lett.250. 377-380 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Hata, J., Yamashita, S., Yagihashi, S., Kato, H., Kabeno, S., Hirai, K., Kuma, K., Kimura, S., Umeki, K., Kotani, T. and Ohtaki, S.: "Stable high level expression of human thyroid peroxidase in cultured chinese hamster ovary cells" Biochem. Biophys. Res. Commun.164. 1268-1273 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yagihashi, S. Matsumoto, K., Kato, H., Hata, J., Kabeno, S., Yamashita, S., Hirai, K., Kuma, K., Umeki, K., Kotani, T. and Ohtaki, S.: "Characterization and application of recombinant human thyroid peroxidase." J. Immunol. Methods.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Yamashita, S., Hata, J., Kabeno, S., Yagihashi, S., Kato, H., Kotani, T. and Ohtaki, S.: "Secretion of membrane binding protein, human thyroid peroxidase, expressed in mammalian cells." J. Endocrinol.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Satoru Yagihashi: "Characterization and application of recombinant human thyroid peroxidase expressed in cultured chinese hamster ovary cells" J.Immunol.Methods.

    • Related Report
      1990 Annual Research Report
  • [Publications] Shinya Yamashita: "Secretion of membrane binding protein,human thyroid peroxidase,expressed in mammalian cells" J.Endocrinol.

    • Related Report
      1990 Annual Research Report
  • [Publications] Kimura,S.: "cDNA-directed expression of human thyroid peroxidase" FEBS Lett.250(2). 377-380 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] Hata,J.: "Stable high level expression of human thyroid peroxidase in cultured chinese hamster ovary cells" Biochem.Biophys.Res.Commun. 164(3). 1268-1273 (1989)

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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